Estrogens and progestins, acting through the estrogen and progesterone receptors (ER and PR) are implicated in the growth and metastases of many breast cancers. Tamoxifen, and other drugs that modify ER and PR action bind in the receptor's ligand binding pocket and lead to development of resistant tumors. We recently developed a novel fluorescence anisotropy microplate assay (FAMA). We used the FAMA to analyze the interaction of liganded ER and PR with their DNA response elements and with a full-length co-activator (SRC1) and demonstrated FAMA's potential for high throughput screening (HTS) assays. We will use the FAMA to develop HTS assays for identification of new classes of ER and PR antagonists targeted at different steps in ER and PR action. The Specific Aims are: 1. We will develop an HTS assay for small molecules that interfere with binding of ER and PR to their respective DNA response elements (EREs and the PRE/GRE) and that exhibit selectivity for ER and PR. 2. We will develop an HTS assay for compounds that interfere with binding of the co-activators SRC1 and Amplified in Breast Cancer 1 (AIB1) to ERE-E2: ER complexes. 3. We will develop HTS assays for small molecules that stimulate the ability of heat shock proteins and chaperones to disassemble complexes of ER and PR with DMA response elements, and with co-activators. HTS suitability studies and assay validation will include analysis of FAMA's precision, quality (Z1 and Z factor), spiking with known inhibitors, and controls for compound fluorescence. For each assay, we will screen approximately 3,000 compounds. Verification assays on candidate and control small molecules will include EMSA, effects on estrogen-dependent growth of breast cancer cells, transient transfections, quantitative RT-PCR of ER and PR-induced cell mRNAs, chromatin IP and RNAi of target proteins. These studies target novel sites for antagonizing cancer cell growth and metastases, and are prototypes for FAMA-based HTS assays.